Protein Tyrosine Phosphatase Gamma (PTPgamma) is a Novel Leukocyte Marker Highly Expressed by CD34 Precursors.

Protein Tyrosine Phosphatase gamma (PTPgamma) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPgamma is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPgamma mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPgamma specific antibody that recognizes the protein by flow cytometry. PTPgamma expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPgamma was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34(+) haematopoietic precursors express high PTPgamma level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPgamma as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation.


Introduction
Protein phosphatases, together with kinases, control critical aspect of cellular signaling as they can modify the phosphorylation level of the cell, leading to pleiotropic effects on proliferation, differentiation and survival (Andersen et al. 2001). Phosphatases can be divided in several families according to their substrate specifi city. Protein tyrosine phosphatases (PTPs) are further divided in two groups. Classical PTPs have phosphotyrosine as exclusive substrate, while dual specifi city phosphatases have a more open active site cleft, allowing them broader substrate specifi city (Tonks, 2003). PTPgamma (PTPγ) is a receptor-like transmembrane protein belonging to the family of classical PTPs; these enzymes can exist in transmembrane (receptor-type PTPs, RPTPs) or non-transmembrane form (described in http://ptp.cshl.edu/). RPTPs can be classifi ed in nine subtypes, according to the combination of structural motifs featuring in the N-terminal moiety; PTPγ belongs to the subtype V, characterized by the presence of a carbonic anhydrase-like and a fi bronectin type III domain at the N-terminus (Barnea et al. 1993). PTPγ has been proposed as a tumor suppressor gene whose expression is lost in various neoplastic diseases including renal cell carcinoma, lung, ovarian, breast and colorectal cancers (LaForgia et al. 1993, Lubinski et al. 1994, Tsukamoto et al. 1992, van Niekerk and Poels, 1999, Liu et al. 2004, Wang et al. 2004, Vezzalini et al. 2007. Involvement in haemopoietic neoplasms has also been reported (van Doorn et al. 2005, Vezzalini et al. 2007. These data suggest a role for PTPγ in the molecular pathways that regulate proliferation and differentiation. Indeed, it has been shown that PTPγ is able to regulate haematopoietic differentiation in a murine embryonic stem cells model (Sorio et al. 1997). We have recently demonstrated that PTPγ is expressed in peripheral blood myeloid and plasmacytoid dendritic cells as well as in monocytes where it is differentially regulated during in vitro differentiation to dendritic cells or macrophages (Lissandrini et al. 2006). These latest fi ndings suggested the possibility that PTPγ represents a novel marker for myeloid cells in the haemopoietic system. Suitable antibodies that can be used in conjunction with lineage-restricted markers are not available. We recently developed a new chicken antibody targeted against the extra cellular domain of PTPγ suitable for fl ow cytometric analysis. Using this newly developed tool, we investigated the expression of this phosphatase in cell lines, peripheral blood samples and purifi ed haemopoietic precursors.

Antibody production
A chicken polyclonal antibody was raised against the sequence CZ NED EKE KTF TKD SDK DLK (residues #390-407 of extra cellular PTPγ sequence); produced IgY were affi nity purifi ed against the same sequence by Aves Labs (Tigard, OR, U.S.A). Pre-immune chicken IgY were collected and purifi ed from the same hen before the immunization process. Briefl y, the antigen was injected into the pectoral muscle of one Americanlaying hen. Three booster injections containing 50 µg of antigen mixed with incomplete Freund's adjuvant were given at 2, 4 and 6 weeks. The eggs were collected daily and stored at 4 °C until the antibodies were extracted. The crude anti-PTPγ chicken IgY (chPTPγ) was further purifi ed using affinity column chromatography against the 20-amino acid peptide. The non-specifi c proteins were washed from the column with washing buffer (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0), until the absorbance at 280 nm decreased to zero and the antibody was then eluted with a desorbing agent (0.1 M glycine, pH 3.0) PTPγ extra cellular domain-enriched supernatants production 293F cells (Invitrogen, Milan, Italy) were transfected with a cDNA encoding for the extra cellular domain of PTPγ (pPTPx), containing the epitope of interest, cloned in pRC/CMV vector (Invitrogen, Milan, Italy). Transfection was achieved using Lipofectamine2000 TM (Invitrogen, Milan, Italy), diluted in OptiMEM TM (Invitrogen, Milan, Italy), according to the manufacturer's optimized protocol for 293F cells. Positive cells were selected by plating transfected samples at about 10 5 cells/mL in RPMI 1640, 10% FBS, 1% Ultraglutamine, 0.9 mg/mL G418 (Invitrogen, Milan, Italy) and incubating them in a humidifi ed atmosphere with 5% CO 2 at 37 °C. Exhaust medium was harvested every 48 hours and centrifuged 5 min at 350 × g to eliminate residual cells. After decanting, the supernatant was further centrifuged for 10 min at 3000 × g to avoid cellular debris. The clarifi ed pools were stored at -20 °C until needed.

RNA extraction and northern blot
10 µg of total RNA was extracted from cell lines using the TRIzol ® reagent (Invitrogen, Milan, Italy) according to the manufacturer instructions and electrophoresed on a 1% formaldehydeagarose gel, blotted onto a Hybond N+ membrane and hybridized to 32 P-labeled cDNA probes prepared by random priming kit (Ready to-go DNA labeling Beads, Amersham Pharmacia Biotech, Uppsala, Sweden) using α-32 P dCTP (ICN, Costa Mesa, CA). The hybridization was performed as described (Sambrook et al. 1989). PTPγ, PTPζ, CD148 and actin cDNA were used as probes. PTPγ cDNA was amplifi ed by PCR from PTPγ pCR ® 3.1 plasmid (Invitrogen) using the following primers: forward 5' CGT CAC CAG TCT CCT 3', reverse 5' GAA GAG GCA GGA GAG 3'. PTPζ cDNA probe was obtained by reverse transcription of total RNA from human astrocytoma tissue and amplifi ed using the following primers: forward 5' CTA GCT GAG GGG TTG GAA TC 3', reverse 5' GTG CCT GTT CTT CCA ACT CC 3'. CD148 cDNA probe was obtained by reverse transcription of total RNA from human peripheral blood granulocytes and amplifi ed using the following primers: forward 5' TGC CAC ACA AGG ACC 3', reverse 5' TGA TTT GCT CCC CAC 3'. The identity of both probes was confi rmed by sequencing. Biotinylated antibodies were detected with streptavidin PE/Cy5 (BioLegend, San Diego, CA). As an isotypic control, pre-immune IgY and matched IgG1-PE or IgG2a-PE (BioLegend, San Diego, CA) were used. Briefl y, purifi ed cells were fi rst blocked with 10% v/v human serum (Invitrogen, Milan, Italy) for 15 minutes at RT, then incubated with chPTPγ (2 µg/10 6 cells) for 1 hour at RT. After one wash with staining buffer (5% FBS in PBS, 0.1% NaN 3 ), cells were stained with goat antichicken Alexa488 (Invitrogen, Milan, Italy) for 30 min at RT and/or with monoclonal antibodies for 15 min at RT. Where biotinylated antibodies were included, a further wash was necessary, followed by an incubation with streptavidin PE/Cy5 for 15 min at RT. Peripheral blood samples were fi rst incubated with 2 µg chPTPγ or pre-immune IgY for 1 hour at RT, then subjected to erythrocytes lysis by addition of 20 volumes of erythrocytes lysis solution (0.886% NH 4 Cl, 0.1% KHCO 3 , 0.006% EDTA) and incubation for 10 min at RT with gentle agitation. After addition of 0.5 volumes of staining buffer and pelleting of cells, a further wash with staining buffer was performed. Cells were then stained with goat anti-chicken Alexa488 for 30 min at RT and/or with monoclonal antibodies as above described. Flow cytometry was performed on a Becton Dickinson FACScan ® fl ow cytometer. Analysis of fl ow cytometry data was performed with FCS Express V3 software (De Novo Software) and geometric mean fl uorescence intensity (MFI) ratio between chPTPγ and isotype control antibodies has been used to evaluate PTPγ density of expression.

PTPγ expression in selected cell lines and antibody validation
The capability to recognize PTPγ by the chicken antibody was evaluated by western blot on the recombinant extra cellular domain. We loaded conditioned media from 293F cells transfected with either an empty vector or with a cDNA for the expression of PTPγ extra cellular domain truncated at the level of the putative transmembrane region. The antibody specifi cally recognize the predicted 120 KD band in the gel lane loaded with the supernatant containing the recombinant extra cellular domain, being the supernatant derived from mock transfected cells negative. A similar, slight background was detectable in both the lanes (Fig. 1A). To validate the use of the antibody for fl ow cytometry we fi rst analyzed PTPγ mRNA expression on a panel of cell lines by northern blot (Fig. 1B) and selected two cellular models: K562 completely lack PTPγ expression and was then transfected with both mock and PTPγ cDNA containing plasmids (K562 γ1). U937 was used as a positive control for native protein. As expected, chPTPγ specifi cally binds to U937 and K562 γ1 while K562 were negative (Fig. 1C).

PTPγ is expressed in peripheral blood leukocytes
We then checked PTPγ expression in peripheral blood leukocytes. Our previous work showed that purifi ed monocytes express PTPγ mRNA, lymphocytes show a very low expression that we interpreted as residual (5%) monocytes contamination of the preparation, while polymorphonucleated cells (PMN) were negative (Lissandrini et al. 2006). Flow cytometry (Fig. 2) confi rmed RNA data for monocytes. However individual staining of both B and T lymphocytes, obtained by CD19 and CD3 double staining, showed that B cells express detectable amount of PTPγ. Surprisingly, PMN showed a slight positive staining for PTPγ in contrast with RNA data. This signal was consistent and led us to hypothesize a residual expression of PTPγ protein on the surface of mature PMN in absence of active synthesis or its expression in a small subpopulation of granulocytes.

PTPγ is expressed in monocytederived dendritic cells (moDC) and further induced by maturation
As moDC expressed high PTPγ levels we wished to confi rm the expression and up-modulation of the protein upon induction of DC maturation by LPS. Cells were obtained from monocytes cultured in presence of IL-4 and GM-CSF and analyzed at day 6 of culture, after 24 h of incubation with or without LPS. In this case, fl ow cytometry with chPTPγ fully confi rmed previous data, showing that moDC express the phosphatase during differentiation (geometric MFI ratio = 4.1) and that the expression is further enhanced upon LPSinduced maturation (geometric MFI ratio = 22.7) (Fig 3A).

PTPγ is strongly expressed in purifi ed CD34 + peripheral blood haematopoietic progenitors and down-modulated during in vitro expansion
We demonstrated that PTPγ expression cause a block in haemopoietic differentiation with an accumulation of cKIT/CD34 + precursors in murine ES cells (Sorio et al. 1997) and, more recently, described high PTPγ expression in endothelial cells (Vezzalini et al. 2007). We therefore wished to examine the presence of PTPγ on CD34 + cells, considered precursors for both haemopoietic and endothelial cells (Suda et al. 2000). We purifi ed CD34 + cells from leukocyte-rich buffy coats and analyzed the expression of this phosphatase. Freshly isolated CD34 + cells showed the highest level of expression detected so far (geometric MFI ratio = 96.7 ± 9.75 SD, n = 4). After a three days treatment with SCF and IL-3 haematopoietic growth factors, known to induce proliferation and differentiation along erythro-myeloid lineage (Zamai et al. 2000), the levels of PTPγ expression decreases (geometric MFI ratio = 4.69 ± 1.50 SD, n = 4) (Fig. 3B).

Discussion
The data here presented are aimed to complement those already exposed in our previous studies (Sorio et al. 1997, Lissandrini et al. 2006) regarding the expression of PTPγ in the haematopoietic system. The chPTPγ antibody recently developed by our group allowed us to move from mRNA expression data to surface labeling of cells and fl ow cytometry analysis. With few exceptions, the data confi rmed at the protein level what had been seen at the RNA level: PTPγ is preferentially expressed by monocytes and dendritic cells. With the sole exception of PMNs the mRNA levels appear to correlate rather well with protein levels, indicating a translational control of the expression levels of this phosphatase. Moreover, its expression levels are substantially modulated when cells are induced to proliferate or differentiate. We also demonstrate that PTPγ is expressed by B cells that represent a class of lymphocytes with the capability to act as antigen-presenting cells together with classic myeloid APCs. CD34 + cells, precursors for both haemopoietic and endothelial cells (Suda et al. 2000), expressed PTPγ indicating that PTPζ is the other member of subtype V receptor type tyrosine phosphatase and is used as a negative control; β-actin is showed for total RNA estimation. As a positive control for PTPζ hybridization we utilized human brain RNA on the same blot (data not shown). C) FACS analysis of K562, K562 transfected with PTPγ full-length cDNA (K562 γ1) and U937 cell lines for PTPγ using chPTPγ antibody. Isotype control staining is showed in gray. undifferentiated progenitors are characterized by expression of this phosphatase. There might be the possibility that PTPγ is required for the maintenance of the undifferentiated phenotype in haemopoietic precursors. This is suggested by previous data in the murine haemopoietic system where its overexpression inhibited erythro-myeloid differentiation with the accumulation of cKIT/CD34 + precursors (Sorio et al. 1997) and by the down modulation following in vitro culture with growth factors here observed. This latter fi nding, in agreement with its proposed tumor suppressor function, would suggest an inverse correlation between haematopoietic proliferation/differentiation and PTPγ expression on haematopoietic progenitor cells. Of course it must be considered that the high expression observed on freshly purifi ed CD34 + cells might also be due to the purifi cation procedure. Of note is the observation that all the anaplastic large cell lymphomas and Reed-Sternberg cells in Hodgkin's disease stained positive for PTPγ expression while only 14% of B cell neoplasms were found positive (Vezzalini et al. 2007). This last result is of note given the expression detected in peripheral blood B cells and suggests different functions associated to specifi c lineages.
We recently found high PTPγ expression in endothelial cells (Vezzalini et al. 2007). The role of PTPγ in vascular biology is completely unknown even if endothelial cells are known to express many transmembrane PTPs involved in the regulation of intercellular contacts of vascular wall cells (Kappert et al. 2005). Our findings suggest a distinct role of PTPγ expression in the haemopoietic system, where its expression is reduced following the initial steps of maturation along the erythro-myeloid series, and endothelial cells where it is found strongly expressed in mature cells.
Altogether our data validate the use of a novel antibody for the detection of PTPγ. The results also indicate that PTPγ has the features of a novel leukocyte marker whose expression is modulated during differentiation and maturation of specifi c leukocyte subsets. Although more work is needed in order to elucidate the biological function of this phosphatase, the evaluation of its expression might represent a useful tool for the characterization of haematopoietic, endothelial and stem cells subsets.